2.1. Alpha (2) Macroglobulin
The α-macroglobulins are members of a protein superfamily of structurally related proteins which also comprises complement components C3, C4 and C5. The human plasma protein alpha (2) macroglobulin (α2M) is a 720 kDa homotetrameric protein primarily known as a proteinase inhibitor and plasma and inflammatory fluid proteinase scavenger molecule (for review see Chu and Pizzo, 1994, Lab. Invest. 71:792). α2M is synthesized as a precursor having 1474 amino acid residues. The first 23 amino acids function as a signal sequence that is cleaved to yield a mature protein with 1451 amino acid residues (Kan et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:2282-2286).
Alpha (2) macroglobulin promiscuously binds to proteins and peptides with nucleophilic amino acid side chains in a covalent manner (Chu et al., 1994, Ann. N.Y. Acad. Sci. 737:291-307) and targets them to cells which express CD91 (also called the α2M receptor or α2MR; Chu and Pizzo, 1993, J. Immunol. 150:48). Binding of α2M to CD91 is mediated by the carboxy-terminal portion of α2M (Holtet et al., 1994, FEBS Lett. 344:242-246) and key residues have been identified (Nielsen et al., 1996, J. Biol. Chem. 271:12909-12912).
Generally known for inhibiting protease activity, α2M binds to a variety of proteases through multiple binding sites (see, e.g., Hall et al., 1981, Biochem. Biophys. Res. Commun. 100(1):8-16). Protease interaction with α2M results in a complex structural rearrangement called transformation, which is the result of a cleavage within the “bait” region of α2M after the proteinase becomes “trapped” by thioesters. The conformational change exposes residues required for receptor binding, allowing the α2M-proteinase complex to bind to the α2MR. Methylamine can induce similar conformational changes and cleavage as that induced by proteinases. The uncleaved form of α2M, which is not recognized by the receptor, is often referred to as the “slow” form (s-α2M). The cleaved form is referred to as the “fast” form (f-α2M) (reviewed by Chu et al., 1994, Ann. N.Y. Acad. Sci. 737:291-307). Recently, it has also been shown that the α2MR can bind to HSPs, such as gp96, hsp90, hsp70, and calreticulin (Basu et al., 2001, Immunity 14(3):303-13).
Studies have shown that in addition to its proteinase-inhibitory functions, α2M, when complexed to antigens, can enhance the antigens' ability to be taken up by antigen presenting cells such as macrophages and presented to T cell hybridomas in vitro by up to two orders of magnitude (Chu and Pizzo, 1994, Lab. Invest. 71:792), and to induce T cell proliferation (Osada et al., 1987, Biochem. Biophys. Res. Commun. 146:26-31). Further evidence suggests that complexing antigen with α2M enhances antibody production by crude spleen cells in vitro (Osada et al., 1988, Biochem. Biophys. Res. Commun. 150:883), elicits an in vivo antibody responses in experimental rabbits (Chu et al, 1994, J. Immunol. 152:1538-1545) and mice (Mitsuda et al., 1993, Biochem. Biophys. Res. Commun. 101:1326-1331). α2M-antigenic peptide complexes have also been shown to induce a cytotoxic T cell response in vivo (Binder et al., 2001, J. Immunol. 166:4698-49720).
2.2. Heat Shock Proteins
Heat shock proteins (HSPs), also referred to as stress proteins, were first identified as proteins synthesized by cells in response to heat shock. Hsps have been classified into five families, based on molecular weight, Hsp100, Hsp90, Hsp70, Hsp60, and smHsp. Many members of these families were found subsequently to be induced in response to other stressful stimuli including nutrient deprivation, metabolic disruption, oxygen radicals, and infection with intracellular or extracellular pathogens (see Welch, 1993, Scientific American 56-64; Young, 1990, Annu. Rev. Immunol. 8:401-420; Craig, 1993, Science 260:1902-1903; Gething et al., 1992, Nature 355:33-45; and Lindquist et al., 1988, Annu. Rev. Genetics 22:631-677).
Heat shock proteins are among the most highly conserved proteins in existence. For example, DnaK, the Hsp70 from E. coli has about 50% amino acid sequence identity with Hsp70 proteins from excoriates (Bardwell et al., 1984, Proc. Natl. Acad. Sci. 81:848-852). The Hsp60 and Hsp90 families also show similarly high levels of intra-family conservation (Hickey et al., 1989, Mol. Cell. Biol. 9:2615-2626; Jindal, 1989, Mol. Cell. Biol. 9:2279-2283). In addition, it has been discovered that the Hsp60, Hsp70 and Hsp90 families are composed of proteins that are related to the stress proteins in sequence, for example, having greater than 35% amino acid identity, but whose expression levels are not altered by stress.
Studies on the cellular response to heat shock and other physiological stresses revealed that the HSPs are involved not only in cellular protection against these adverse conditions, but also in essential biochemical and immunological processes in unstressed cells. HSPs accomplish different kinds of chaperoning functions. For example, members of the Hsp70 family, located in the cell cytoplasm, nucleus, mitochondria, or endoplasmic reticulum (Lindquist et al., 1988, Ann. Rev. Genetics 22:631-677), are involved in the presentation of antigens to the cells of the immune system, and are also involved in the transfer, folding and assembly of proteins in normal cells. HSPs are capable of binding proteins or peptides, and releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or low pH.
2.3. Immunogenicity of HSP-Peptide Complexes
Srivastava et al. demonstrated immune response to methylcholanthrene-induced sarcomas of inbred mice (1988, Immunol. Today 9:78-83). In these studies, it was found that the molecules responsible for the individually distinct immunogenicity of these tumors were glycoproteins of 96 kDa (gp96) and intracellular proteins of 84 to 86 kDa (Srivastava et al., 1986, Proc. Natl. Acad. Sci. USA 83:3407-3411; Ullrich et al., 1986, Proc. Natl. Acad. Sci. USA 83:3121-3125). Immunization of mice with gp96 or p84/86 isolated from a particular tumor rendered the mice immune to that particular tumor, but not to antigenically distinct tumors. Isolation and characterization of genes encoding gp96 and p84/86 revealed significant homology between them, and showed that gp96 and p84/86 were, respectively, the endoplasmic reticular and cytosolic counterparts of the same heat shock proteins (Srivastava et al., 1988, Immunogenetics 28:205-207; Srivastava et al., 1991, Curr. Top. Microbiol. Immunol. 167:109-123). Further, Hsp70 was shown to elicit immunity to the tumor from which it was isolated but not to antigenically distinct tumors. However, Hsp70 depleted of peptides was found to lose its immunogenic activity (Udono and Srivastava, 1993, J. Exp. Med. 178:1391-1396). These observations suggested that the heat shock proteins are not immunogenic per se, but form noncovalent complexes with antigenic peptides, and the complexes can elicit specific immunity to the antigenic peptides (Srivastava, 1993, Adv. Cancer Res. 62:153-177; Udono et al., 1994, J. Immunol., 152:5398-5403; Suto et al., 1995, Science, 269:1585-1588).
Noncovalent complexes of HSPs and peptide, purified from cancer cells, can be used for the treatment and prevention of cancer and have been described in PCT publications WO 96/10411, dated Apr. 11, 1996, and WO 97/10001, dated Mar. 20, 1997 (U.S. Pat. No. 5,750,119 issued Apr. 12, 1998, and U.S. Pat. No. 5,837,251 issued Nov. 17, 1998, respectively, each of which is incorporated by reference herein in its entirety). The isolation and purification of stress protein-peptide complexes has been described, for example, from pathogen-infected cells, and can be used for the treatment and prevention of infection caused by the pathogen, such as viruses, and other intracellular or extracellular pathogens, including bacteria, protozoa, fungi and parasites (see, for example, PCT Publication WO 95/24923, dated Sep. 21, 1995). Immunogenic stress protein-peptide complexes can also be prepared by in vitro complexing of stress protein and antigenic peptides, and the uses of such complexes for the treatment and prevention of cancer and infectious diseases has been described in PCT publication WO 97/10000, dated Mar. 20, 1997 (U.S. Pat. No. 6,030,618 issued Feb. 29, 2000. The use of stress protein-peptide complexes for sensitizing antigen presenting cells in vitro for use in adoptive immunotherapy is described in PCT publication WO 97/10002, dated Mar. 20, 1997 (see also U.S. Pat. No. 5,985,270 issued Nov. 16, 1999).
2.4. The Alpha (2) Macroglobulin Receptor, or “CD91”
The alpha (2) macroglobulin receptor (herein referred to interchangeably as either “α2MR” or “the α2M receptor”), also known as LDL (low-density lipoprotein) receptor-Related Protein (“LRP”) or CD91, is primarily expressed in liver, brain and placenta. The α2M receptor is a member of the low density lipoprotein receptor family. The extracellular domain of the human receptor comprises six 50-amino acid EGF repeats and 31 complement repeats of approximately 40-42 amino acids. The complement repeats are organized, from the amino to the carboxy-terminus, into clusters of 2, 8, 10 and 11 repeats, called Cluster I, II, III and IV (Herz et al., 1988, EMBO J. 7:4119-4127). One study points to Cluster II (C1-II), which contains complement repeats 3-10 (CR3-10), as the major ligand binding portion of the receptor (Horn et al., 1997, J. Biol. Chem. 272:13608-13613). The α2M receptor plays a role in endocytosis of a diversity of ligands. In addition to α2M, other ligands of α2MR include lipoprotein complexes, lactoferrin, tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and exotoxins. Additional examples of ligands of of/MR can be found in PCT publication WO 97/04794 and U.S. Pat. No. 6,156,311. Thus, the α2M receptor plays roles in a variety of cellular processes, including endocytosis, antigen presentation, cholesterol regulation, ApoE-containing lipoprotein clearance, and chylomicron remnant removal.
Human α2M is synthesized as a 1474 amino acid precursor, the first 23 of which function as a signal sequence that is cleaved to yield a 1451 amino acid mature protein (Kan et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:2282-2286). In experiments with recombinant protein, the carboxy-terminal 138 amino acids of α2M (representing amino acids 1314-1451 of the mature protein) was found to bind the receptor. This domain has been called the RBD (receptor-binding domain; Salvesent et al., 1992, FEBS Lett. 313:198-202; Holtet et al., 1994, FEBS Lett. 344:242-246). An RBD variant (RBDv), a proteolytic fragment of α2M comprising an additional 15 amino terminal residues (representing amino acids 1314-1451 of the mature protein) binds to the receptor with almost the same affinity as α2M-proteinase (Holtet et al., 1994, FEBS Lett. 344:242-246).
Alignment of α2MR ligands identifies a conserved domain present in the RBDs of α macroglobulins. The conserved sequence spans amino acids 1366-1392 of human α2M. Conserved residues within this domain are Phe1366, Leu1369, Lys1370, Val1373, Lys1374, Glu1377, Val1382, Arg1384 (Nielsen et al., 1996, J. Biol. Chem. 271:12909-12912). Of these, Lys1370 and Lys1374 were shown to be critical for receptor binding (Nielsen et al., 1996, J. Biol. Chem. 271:12909-12912).
Binding of ligands, including the binding to α2M, to α2MR is inhibited by α2MR-associated protein (RAP). RAP is a 39 kDa folding chaperone that resides in the endoplasmic reticulum and is required for the normal processing of α2MR. RAP has the ability to competitively inhibit the binding of all α2MR to all α2MR ligands tested. One study shows RAP to bind to complement repeats C5-C7 in cluster II (C1-II) of α2MR (Horn et al., 1997, J. Biol. Chem. 272:13608-13613); another shows RAP to bind to all two complement repeat-modules in C1-II except the C9-C10 module (Andersen et al., J. Biol. Chem., Mar. 24, 2000, PMID: 10747921; published electronically ahead of print). Three structural domains, 1, 2 and 3, have been identified in RAP, consisting of amino acid residues 18-112, 113-218 and 219-323, respectively. Ligand competition titration of recombinant RAP domains indicates that determinants for the inhibition of test ligands reside in the C-terminal regions of domains 1 and 3 (Ellgaard et al., 1997, Eur. J. Biochem. 244:544-51).
The use of CD91 as a heat shock protein receptor, cells that express CD91 bound to an HSP, antibodies and other molecules that bind a CD91-HSP complex, screening assays to identify compounds that modulate the interaction of an HSP with CD91, methods for using compositions comprising CD91, and CD91 sequences for the diagnosis and treatment of immune disorders, proliferative disorders, and infectious diseases have also been described in PCT publication WO 01/92474, dated Dec. 6, 2001. Complexes of alpha (2) macroglobulin associated with antigenic molecules for use in immunotherapy and methods for using such compositions in the diagnosis and treatment of proliferative disorders, and infectious diseases have also been described in PCT publication WO 01/91787 dated Dec. 6, 2001. Binder et al. showed that in vitro reconstituted complexes of alpha (2) macroglobulin and antigenic peptides elicit specific CTL response (Binder et al, 2001, J. Immunol. 166:4968-4972).
2.5. Antigen Presentation
Major histocompatibility complex (MHC) molecules present antigens on the cell surface of antigen-presenting cells. Antigens are processed by two distinct antigen processing routes depending upon whether their origin is intracellular or extracellular. Intracellular or endogenous protein antigens, i.e., antigens synthesized within the antigen-presenting cell, are presented by MHC class I (MHC I) molecules to CD8+ cytotoxic T lymphocytes. On the other hand, extracellular or exogenously synthesized antigenic determinants are presented on the cell surface of “specialized” or “professional” APCs (macrophages, for example) by MHC class II molecules to CD4+ T cells (see, generally, Fundamental Immunology, W. E. Paul (ed.), New York: Raven Press, 1984). This compartmental segregation of antigen processing routes is important to prevent tissue destruction that could otherwise occur during an immune response as a result of shedding of neighboring cell MHC I antigens.
The heat shock proteins chaperone a wide array of peptides, depending upon the source from which the HSP is isolated (for review, see Srivastava et al., 1998, Immunity 8: 657-665). Tumor-derived HSP carries tumor-antigenic peptides (Ishii et al., 1999, J. Immunology 162:1303-1309); gp96 preparations from virus-infected cells carry viral epitopes (Suto and Srivastava, 1995, Science 269:1585-1588; Nieland et al., 1996, Proc. Natl. Acad. Sci. USA 95:1800-1805), and gp96 preparations from cells transfected with model antigens such as ovalbumin or β-galactosidase are associated with the corresponding epitopes (Arnold et al., 1995, J. Exp. Med. 182:885-889; Breloer et al., 1998, Eur. J. Immunol. 28:1016-1021). The association of gp96 with peptides occurs in vivo (Menoret and Srivastava, 1999, Biochem. Biophys. Research Commun. 262:813-818). HSP-peptide complexes, whether isolated from cells (Tamura et al., 1997, Science 278:117-120), or reconstituted in vitro (Blachere et al., 1997, J. Exp. Med. 186:1183-1406) are excellent immunogens and have been used extensively to elicit CD8+ T cell responses specific for the HSP-chaperoned antigenic peptides.
The capacity of HSP-peptide complexes to elicit an immune response is dependent upon the transfer of the peptide to MHC class I molecules of antigen-presenting cells (see for example: Suto and Srivastava, 1995, supra). Endogenously synthesized antigens chaperoned by gp96 in the endoplasmic reticulum [ER] can prime antigen-specific CD8+ T cells (or MHC I-restricted CTLs) in vivo; this priming of CD8+ T cells requires macrophages. However, the process whereby exogenously introduced gp96-peptide complexes elicit the antigen-specific CD8+ T cell response is not completely understood since there is no established pathway for the translocation of extracellular antigens into the class I presentation machinery. Yet antigenic peptides of extracellular origin associated with HSPs are somehow salvaged by macrophages, channeled into the endogenous pathway, and presented by MHC I molecules to be recognized by CD8+ lymphocytes (Suto and Srivastava, 1995, supra; Blachere et al., 1997, J. Exp. Med. 186:1315-22).
2.6. HSP-CD91 Interactions
The studies reported by Basu et al. indicate that the heat shock proteins gp96, hsp90, hsp70, and calreticulin are additional ligands for the CD91 (Basu et al., 2001, supra). Gp96 engages a region of CD91, located in an amino terminal fragment termed the p80 fragment (Binder et al, 2000, Nature immunology, 1:151-155; WO 01/92474). The human gp96-coding gene has been mapped previously by us at chromosome 12 (q24.2 q24.3) (Maki et al., 1993, Somatic Cell Mol. Gen. 19:73-81). It is of interest in this regard that the CD91 gene has been mapped to the same chromosome and at a not too distant location (q13 q14) (Hilliker et al. Genomics 13:472-474). Gp96 binds CD91 directly and not through other ligands such as α2M. Homogenous preparations of gp96, in solution, or cross-linked to a solid matrix, bind to the CD91. Indeed, the major ligand for the CD91, α2M, actually inhibits interaction of gp96 with CD91, instead of promoting it, providing evidence that gp96 is a direct ligand for the CD91. The 80 kDa protein, p80, shown to bind gp96 is clearly an amino terminal degradation product of the a subunit of the CD91 (Binder et al, 2000, Nature immunology, 1:151-155). Degradation products of the CD91 in this size range have also been observed in previous studies (Jensen et al., 1989, Biochem. Arch. 5:171-176), and may indicate the existence of a discrete ectodomain in the CD91 which may be particularly sensitive to proteolytic cleavage.
The observations of Basu et al. that α2 macroglobulin and anti-CD91 antibodies inhibit re-presentation by each of the four HSPs completely, indicate that CD91 is the only receptor for the 4 HSPs (Basu et al., 2001, supra). Considering the increasingly obvious role which the HSPs play in innate (Basu et al., 2000, Int. Immunol. 12(11):1539-1546) and adaptive immune response, this observation is somewhat counter-intuitive. However, the data on complete inhibition by two independent means are quite compelling (PCT publication WO 01/92474, dated Dec. 6, 2001). Binder reported significant differences between hsp70 and hsp90/gp96 in their ability to compete for binding to gp96 receptors (Binder et al., 2000, J. Immunol. 165:2582-2587). Another group has also observed similar differences between gp96 and hsp70 (Arnold-Schild et al., 1999, 162:3757-3760). These differences are not inconsistent with Basu's report pointing to a single receptor for the 4 HSPs. They simply suggest that the various HSPs interact with a single receptor with widely differing affinities
As shown in Binder et al., the heat shock protein-CD91 interaction provides a new type of function for CD91, or a fragment thereof, a function of a sensor, not only of the extracellular environment with its previously known plasma-based ligands, but also a sensor of the intracellular milieu as well. HSPs such as gp96 are obligate intracellular molecules and are released into the extracellular milieu only under conditions of necrotic (but not apoptotic) cell death (PCT publication WO 01/92474, dated Dec. 6, 2001). Thus, the CD91 may act as a sensor for necrotic cell death, just as the scavenger receptor CD36 and the recently identified phosphatidyl serine-binding protein act as sensors of apoptotic cell death and receptors for apoptotic cells (Savill et al., 1992, J. Clin. Invest. 90:1513-1522; Fadok et al., 2000, Nature 405:85-90). Interaction of the macrophages with the apoptotic cells leads to a down-regulation of the inflammatory cytokines such as TNF (Fadok et al., 2000, supra), while gp96-APC interaction leads to re-presentation of gp96-chaperoned peptides by MHC I molecules of the APC, followed by stimulation of antigen-specific T cells (Suto and Srivastava, 1995, supra) and, in addition, secretion of pro-inflammatory cytokines such as TNF, GM-CSF and IL-12. Interestingly, α2M, an independent ligand for the CD91, inhibits representation of gp96-chaperoned peptides by macrophages. This observation of Binder suggests that re-presentation of gp96-chaperoned peptides can not occur physiologically in blood, but only within tissues as a result of localized necrotic cell death. This is consistent with the complete absence of gp96 or other HSPs in blood under all conditions tested. Together, Binder's observations point towards a possible mechanism whereby the release of HSPs in the blood as a result of severe tissue injury and lysis will not lead to a systemic and lethal pro-inflammatory cytokine cascade.
It is possible, therefore, that CD91 renders it possible for the APCs to sample (i) the extracellular milieu of the blood through α2M and other plasma ligands and (ii) the intracellular milieu of the tissues through HSPs, particularly of the gp96 family. The former permits APCs to implement their primordial phagocytic function, while the latter allows them to execute its innate and adaptive immunological functions. Viewed in another perspective, recognition of apoptotic cells by APCs through CD36 or phophatidyl serine, leads to anti-inflammatory signals, while interaction of the APC with necrotic cells through CD91 leads to pro-inflammatory innate and adaptive immune responses (see Srivastava et al., 1998, Immunity 8: 657-665).
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.